Hybridization has traditionally been used to describe the general technique by which complementary strands of deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules or combinations of DNA and RNA are separated into single strands and then allowed to renature or reanneal and reform base-paired double helices. Hybridization techniques are generally classified in three major classes: solution hybridization whereby the individual cell is disrupted and the internal nucleic acid is extracted into solution prior to hybridization; this technique includes different levels of purification of the nucleic acid prior to hybridization; filter or blot hybridization, whereby DNA or RNA is bound to a solid matrix either directly or after separation of the individual nucleic acid fragments on e.g. an agarose gel or a polyacrylamide gel and the subsequent hybridization is carried out with a labelled probe for detection of hybrids; and in situ hybridization (ISH) whereby a method is provided for the detection and localization of specific nucleic acid sequences directly in a specific structure, e.g. within a cell, a tissue, a nucleus or a chromosome. Although these techniques are based on the specific interaction between the target and a probe capable of binding specifically to the sequence to be detected, these techniques are quite different and distinguishable from each other.
In situ hybridization allows detection of specific cellular or chromosomal nucleic acid sequences in the cellular material such as in paraffin-embedded tissue sections, fresh or frozen biopsies, cells or chromosomes. Traditionally, nucleic acid probes having a base sequence that is sufficient complementary to the target sequence to be detected have been used. For this type of detection, nucleic acid probes labelled with detectable groups other than radioisotopes have been used widely.
The demand for more sensitive methods is increasing. Ways of increasing the sensitivity are to use an enhancing detection system and/or to increase the number of probes with different sequences targeting the nucleic acid sequences to be detected. When closely related sequences are to be distinguished, it is often limited how many probe variations can be used to detect a given target.
An increase of the sensitivity for in situ nucleic acid hybridization in tissue sections or cell smears has been described using the so-called "in situ PCR". The principle of in situ PCR is to combine the techniques of PCR and ISH through the amplification of specific nucleic acid sequences inside the individual cells, resulting in an increase of copy numbers to levels detectable by ISH. Reproducible results depend on the integrity of the sample. During pretreatment of the sample, the cell membrane may be damaged giving rise to a risk of so-called "diffusion artefacts". PCR products may leak out of cells and serve as template for extracellular amplification. This may give rise to false positive signals. A recent report summerizes the pitfalls of "in situ PCR" (Cell Vision, 3, 231-235 (1995)).
In accordance with the present invention binding partners and protocols are provided for in situ hybridization procedures for the detection of specific nucleic acid sequences in a sample of eucaryotic origin. In the present method, hybridization is performed using a binding partner, which is a polymeric strand of polymerized moieties having a non-cyclic backbone, the polymeric strand being capable of hybridizing to the nucleic acid sequence to be determined. Examples of such polymeric strands are given in WO 92/20702.
In WO 92/20702, the term Peptide Nucleic Acid (PNA) was introduced to describe some compounds having a non-cyclic backbone and nucleic acid binding properties.
In WO 93/24652, PNA probes have been postulated to be feasible for in situ hybridization carried out without addition of a denaturing agent, such as formamide, and further with hybridization performed at low temperature. The postulated simplification of the in situ hybridization procedure is based on the prior assumption that PNA form triplex structures with double-stranded DNA. In WO 93/24652, there is no experimental data to support this simplification. It has now been shown that the hypothesis of triplex formation put forward in 1991 (Science, 254, 1497-1500 (1991)) only applies if a homopyrimidine PNA is combined with a homopurine DNA whereby (PNA).sub.2 /DNA triplexes are formed, such as (PNA(T)).sub.2 (polydA) (TIBTECH, 11, 384-386 (1993)).